Abstract The ubiquitin system controls many cellular pathways. Proteins are tagged with ubiquitin via an E1-E2-E3 cascade, and this frequently leads to degradation via the proteasome. Deubiquitinating enzymes (Dubs) act to remove ubiquitin from proteins, leading to reversal of the signaling input generated by the ubiquitination event. A small number of Dubs have been linked to important cellular pathways linked to transcriptional control, oncogenesis, tumor suppression, and neurodegeneration, yet the biological functions, interaction partners, and substrates of the majority of Dubs are unknown. We have recently developed a new informatic and experimental platform for semi high-throughput proteomic analysis of protein complexes and have applied this technology to 75 of the 95 Dubs encoded by the human genome, using 293T cells as an initial cell system. These studies reveal that a substantial fraction of Dubs are stably associated with previously identified and novel protein complexes. In many cases, the identity of associated proteins provides the first indications of the biological functions or pathways in which uncharacterized Dubs may operate. Several Dubs and/or their associated proteins have been identified in shRNA-based checkpoint screens, providing candidate biological pathways for many Dubs and their newly identified complexes. Several themes have emerged: 1) Dubs are frequently components of large molecular machines, 2) Dubs frequently associate with E3s, and 3) WD40- repeat containing proteins (including WD40 proteins that have recently been shown to bind ubiquitin) are frequently associated with Dubs, which is interesting in light of the recent finding that WDR48 activates Usp1. Our approach identified many tightly associated proteins but known substrates of a small number of well- studied Dubs were not identified, suggesting that alternative approaches are required to identify more weakly bound substrates of Dubs. This proposal seeks to further elucidate the functions and pathways controlled by Dubs. In aim 1,we will complete our systematic analysis of the Dub proteome, and will examine the proteome of substrate-trapping Dub mutants with the goal of identifying weakly bound substrates. Further enhancement and integration of our informatics platform and database is also proposed. Aim 2 has the broad goal of elucidating emerging themes in the function and regulation of Dubs, including: 1) the role of WD40 proteins as activator and substrate receptor subunits of Dubs, and 2) the cross-regulation of Dubs, E3s, and targets of E3s. These themes will be addressed using 2 specific Dub networks we have identified via our proteomics analysis. Together, these experiments will provide a powerful resource for the field devoted to elucidating the functions and targets of Dubs and will begin to uncover important regulatory networks that appear to be commonly employed to regulate Dub function.